The endothelium forms the main barrier to the passage of macromolecules and circulating cells from blood to tissues. Endothelial permeability is in large part regulated by intercellular junctions. These are complex structures formed by transmembrane adhesive molecules linked to a network of cytoplasmic/cytoskeletal proteins. At least four different types of endothelial junctions have been described: tight junctions, gap junctions, adherence junctions and syndesmos. These organelles have some features and components in common with epithelial cells but there are also some that are specific for the endothelium. The mechanisms that regulate the opening and closing of endothelial junctions are still obscure. It is conceivable that inflammatory agents increase permeability by binding to specific receptors generating intracellular signals, which in turn cause cytoskeletal reorganization and opening of interendothelial cell gaps. Endothelial junctions also control leukocyte extravasation. Once leukocytes have adhered to the endothelium, a coordinated opening of interendothelial cell junctions occurs. The mechanism by which this takes place is unknown, but it might present characteristics similar to that triggered by soluble mediators.

Abstract-Endothelial adherens junctions (AJ) promote intercellular adhesion and may contribute to the control of vascular permeability. These structures are formed by a transmembrane and cell-specific adhesive protein, vascular endothelial (VE)-cadherin, which is linked by its cytoplasmic tail to intracellular proteins called catenins (␣-catenin, ␤-catenin, and plakoglobin) and to the actin cytoskeleton. Little is known about the functional regulation of AJ in endothelial cells. In this study, we analyzed the effect of histamine on AJ organization in cultured endothelial cells. We first observed that histamine induced detectable intercellular gaps only in loosely-confluent cells, whereas this effect was strongly reduced or absent in long-confluent cultures. Despite this difference, in vitro permeability was augmented by histamine in both conditions. In resting conditions, tyrosine phosphorylation of AJ components and permeability values were higher in recently-confluent cells as compared with long-confluent cells. Histamine did not affect the phosphorylation state of AJ in recently-confluent cells but strongly increased this parameter in long-confluent cultures. In addition, in long-confluent cells, histamine caused dissociation of VE-cadherin from the actin cytoskeleton measured by a decrease of the amount of the molecule in the detergent-insoluble fraction of the cell extracts. Dibutyryl cAMP was able to prevent the effect of histamine on both tyrosine phosphorylation of AJ components and on endothelial permeability. The effect of histamine was specific for VE-cadherin because the phosphorylation state of neural (N)-cadherin, the other major endothelial cadherin, was unchanged by this agent. Hence AJ components are a target of histamine activation cascade; we suggest that induction of tyrosine phosphorylation of VE-cadherin and catenins contributes to the histamine effect on permeability, even in absence of frank intercellular gaps and cell retraction. (Arterioscler Thromb Vasc Biol. 1999;19

In vivo, intact endothelium presents a low turnover rate, however, when junctions are disrupted cells gain the capacity to migrate and proliferate. This capacity is then lost when cell to cell contacts are reorganized (4).Endothelial cell junction components are therefore good candidates for transferring migration and growth inhibitory signals. Previous work (5) showed that protein membrane extracts from confluent endothelial cells were able to inhibit the growth of sparse endothelium but not of other cell types, suggesting the existence of membrane associated endothelial growth inhibitory proteins.It was previously found that endothelial cells express a cell specific member of the cadherin family (cadherin-5 or vascular endothelial cadherin [VE-cadherin]) 1 (6)(7)(8). This molecule is so far the only cadherin consistently organized at interendothelial adherence junctions (8-10). VE-cadherin is a constitutive component of all types of endothelia (8). As the other members of the family (11-15), VE-cadherin has adhesive properties and mediates homotypic cell adhesion (16). The intracellular domain interacts with cytoplasmic proteins called catenins (17) that transmit the adhesion signal and contribute to the anchorage of the protein to the actin cytoskeleton (18,19).In other types of tissues, cadherins can act as tumor suppressors. Reduced cadherin expression and/or activity is associated with enhanced tumor cell invasive potential and loss of differentiated characteristics (11)(12)(13)(14)(15)(18)(19)(20)(21)(22)(23). In agreement with these observations, VE-cadherin expression was found to be strongly reduced in angiosarcomas (24).In this paper we investigated the effect of VE-cadherin on cell growth. The results reported show that VE-cadherin can indeed transfer growth negative signals to the cells. This molecule can therefore contribute to density dependent inhibition of endothelial cell growth. MethodsCells. Human endothelial cells from umbilical vein (HUVEC) were isolated and cultured in M199 and 20% NCS (newborn calf serum) as previously described (8). Chinese hamster ovary (CHO) cells and mouse connective tissue fibroblast L929 cells were obtained from the American Tissue Type Collection and cultured in DMEM with 10% FCS (both from GIBCO, Life Technologies, Paisley, U.K.) (16). Full length VE-cadherin cDNA was cloned from human endothelial cells and inserted into pECE eukaryotic expression vector. CHO and L929 cells were cotransfected with pECE-VE-cadherin construct and pSV 2 neo plasmids by calcium phosphate precipitation as described (16). Control cells were transfected with empty pECE and pSV 2 neo plasmids, selected, cloned, and cultured as VE-cadherin transfectants (16). VE-cadherin expression in the clones used was comparable with that of HUVEC by Western and Northern blot analysis (16).For transfection of CHO cells with truncated VE-cadherin, full length VE-cadherin cDNA cloned in pBluescript vector (16)